Corneal epithelial stem cell disease or dysfunction, known as limbal stem cell deficiency, is among the most common blinding eye conditions worldwide. There was no effective therapy for these conditions until the potential of limbal transplantation for surface reconstruction of eyes with this condition was realized in the past decade. The success of limbal transplantation has been attributed to the healing potential of the corneal epithelial stem cells that are only a small subpopulation contained within the mixture of cells transplanted in these grafts. The ability to isolate a pure population of corneal epithelial stem cells from small limbal biopsies, expand them in culture and use them for regenerating a corneal surface of normal phenotype and regenerative capacity would represent a major advance in this field. But to date, no specific markers for corneal epithelial stem cells have been identified. Isolation of corneal epithelial stem cells has not been achieved. Our preliminary studies have provided encouraging results in support of our hypothesis that the novel isolation methods and proposed markers for stem cells and basal cells in non-ocular tissues can be utilized to isolate a pure population of corneal epithelial stem cells. Two proposed Specific Aims will realize the long-term objectives. Aim 1 will create novel approaches to isolate corneal epithelial stem cells based on their unique properties and molecular markers for use in corneal tissue engineering. Three isolating strategies in combination will be used: 1) Size sorting based on the correlation between cell differentiation and enlarged size; 2) Enrichment by their rapid adherence to extracellular matrix based on their higher-expression of beta1integrin; 3) Purification of stem cells as a side population by flow cytometry using a vital DNA binding dye, Hoechst 33342. Aim 2 will search for new specific markers for corneal epithelial stem cells by characterizing gene expression patterns in the purified populations of stem cells using advanced gene array/microarray techniques. With the proposed Aims accomplished, pure populations of corneal epithelial stem cells will be available for the first time, which will make the corneal epithelial stem cell concept become reality and bring stem cell research and clinical application into a new age. This work will have important scientific significance and high impact on the adult stem cell concept, not only for cornea and ocular surface, but also for other tissues.